Optimization of Agrobacterium-mediated transformation in spring bread wheat using mature and immature embryos
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Wheat is the most widely grown staple food crop in the world and accounts for dietary needs of more than 35% of the human population. Current status of transgenic wheat development is slow all over the world due to the lack of a suitable transformation system. In the present study, an efficient and reproducible Agrobacterium-mediated transformation system in bread wheat (Triticum aestivum L.) is established. The mature and immature embryos of six recently released high yielding spring bread wheat genotypes were used to standardize various parameters using Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA3301 having gus and bar as marker genes. The optimum duration for embryo pre-culture, inoculation time and co-cultivation were 2 days, 30 min and 48 h, respectively. The bacterial inoculum concentration of OD of 1 at 600 nm showed 67.25% transient GUS expression in the histochemical GUS assay. The filter paper based co-cultivation limits the Agrobacterium overgrowth and had 82.3% explants survival rate whereas medium based strategy had 22.7% explants survival only. The medium having picloram 4 mg/l along with antibiotics (cefotaxime 500 mg/l and timentin 300 mg/l) was found best suitable for initial week callus induction. The standardized procedure gave overall 14.9% transformation efficiency in immature embryos and 9.8% in mature embryos and confirmed by gene-specific and promoter-specific PCR and southern analysis. These results indicate that the developed Agrobacterium-mediated transformation system is suitable for diverse wheat genotypes. The major obstacle for the implication of the CRISPR-based genome editing techniques is the non-availability of a suitable transformation system. Thus, the present system can be exploited to deliver the T-DNA into the wheat genome for CRISPR-based target modifications and transgene insertions.
KeywordsWheat, Triticum aestivum, cereal, Agrobacterium bar gene Histochemical GUS assay Southern analysis Transformation
We are highly thankful to Dr. Viswanathan Chinnusamy, Dr. Santosh Kumar, IARI, New Delhi and Dr. Monika Dalal, NRCPB, New Delhi for extending their guidance and facility for southern analysis.
MHM conceived the project and designed the experiments with RK and AK. RK, AK and DS conducted experiments. RK, AK, and KV wrote the manuscript. GPS provides necessary infrastructure and facilities to conduct various experiments. All authors read and approved the final manuscript.
This work is financially supported by the Indian Council of Agricultural Research, New Delhi, India, under the project entitled “ICAR Network Project on Functional Genomics and Genetic Modification in Crops (NPFGGM)” (Project No. 1006474).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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